Linked-fragment sequencing

Abstract

The invention generally relates to sequencing library preparation methods. In certain embodiments, two or more template nucleic acids are joined together by a linking molecule, such as a PEG derivative. Identical copies of a nucleic acid fragment or both strands of a duplex fragment may be linked together. The linked nucleic acids are amplified, creating linked amplicons. Emulsion PCR with linked primers creates linked template nucleic acids for seeding sequencing clusters and errors can be readily identified by their presence on only one of the linked fragments.

What is claimed is: 
     
       1. A method of sequencing a nucleic acid, the method comprising:
 ligating adapters to a sense strand and an antisense strand of a duplex nucleic acid fragment to create ligation products wherein at least one of the adapters is a linking adapter comprising a Y-adapter having a duplex portion and a varying stem region with a primer linked to one strand of the varying stem region, said primer being complementary to a portion another strand of the varying stem region; 
 extending the primer with a strand displacing polymerase to prepare extended ligation products comprising joined copies of the sense strand of the duplex nucleic acid fragment and the antisense strand of the duplex nucleic acid fragment; 
 loading the extended ligation products on a sequencing flow cell; and 
 sequencing the extended ligation products. 
 
     
     
       2. The method of  claim 1 , wherein the linker is selected from the group consisting of a polyethylene glycol derivative, an oligosaccharide, a lipid, a hydrocarbon, a polymer, an inverted base, and a protein. 
     
     
       3. The method of  claim 1 , wherein the linking adapter comprises streptavidin and biotinylated DNA. 
     
     
       4. The method of  claim 3 , wherein the linking adapter comprises a streptavidin coated bead. 
     
     
       5. The method of  claim 1 , wherein the each extended ligation product forms a seed for a single cluster. 
     
     
       6. The method of  claim 1 , wherein sequencing the extended ligation products comprises a first sequencing read of the sense strand copies and a second sequencing read of the antisense strand copies, the method further comprising identifying a consensus base call by comparing the first and second sequencing reads.